The Epigenetic Regulation of Blinatumomab Gene Expression: Tumor Cell-dependent T cell Response against Lymphoma Cells and Cytotoxic Activity

Conventional treatment for cancer such as surgical resection and chemotherapy can cause damage in cases with advanced cancers. Moreover, the identification of tumor-specific targets has great importance in T-cell therapies. For decades, T cell activity has been stimulated to improve anti-tumor activity. Bispecific antibodies have attracted strong interest from pharmaceutical companies, for their diagnostic and therapeutic use. Blinatumomab is a first-in-class bispecific T engager antibody for the treatment of relapsed or refractory precursor B- cell acute lymphoblastic leukemia. But, it can benefit several cases with CD19+ malignancies in the future. PhiC31 integrase-based vectors could selectively integrate therapeutic transgenes into pseudo-attP sites in CHO genome. In this study, production of Blinatumomab in CHO cells using this type of vectors was investigated. We evaluated the effects of histone deacetylases (HDACs) inhibitors such as sodium butyrate and valproic acid, on specific productivity and cell viability of antibody expressing cells. Although sodium butyrate increased specific productivity about 1.7-fold and valproic acid about 1.4-fold, valproic acid was found more efficient because of its less cytotoxic effect on cell growth. We examined the efficacy of expressed Blinatumomab at various effector to target (E/T) ratios. A dose-response analyses of calcein-acetoxymethyl release assay illustrated that the effective dose of expressed mAb required for antibody mediated cytotoxicity was 100 ng/ml and the expressed mAb was more effective at E/T ratios of 10:1 and 5:1. Results of this study indicated that the expressed blinatumomab can be useful for enhancing the cytotoxicity of CD3+ T-cells against CD19 + target cells in vitro.

plasmid into pseudo attP site in mammalian genomes (2). PhiC31 integrase system is considered as a specific tool for gene therapy (3,4) and transgenic research (2,5). The efficiency of phiC31-integrase has been indicated to be comparable with that of the widely used Cre/loxP system. Furthermore, flippase (FLP) recombinase shows only 10% recombination activity on chromosomal targets in comparison with Cre recombinase (6). Cre and FLP cause deletion of the gene after integration (7) whereas phiC31 integrase can catalyze unidirectional and irreversible recombination between attB and pseudo attP sites (3). Development of phiC31 integrase-based vectors for prolonged therapeutic gene expression, demonstrated that it is a robust and reliable gene delivery system (4,8). Sodium butyrate (NaBut) treatment increases the specific productivity of recombinant proteins in mammalian cells; but, it declines cell growth and can provoke apoptosis (9).
NaBut inhibits the activity of many histone deacetylases, induces hyperacetylation of histones.
Histone acetylation could modify chromatin structure, lead to transcription factors and polymerases binding as well as improving gene expression (10). Due to its impact on epigenetic mechanisms, NaBut has attracted many interest for the prevention and treatment of different diseases such as genetic/metabolic conditions and neurological degenerative disorders (11). Valproic acid (VPA), a histone deacetylase inhibitor (HDACi), can cause impaired epigenetic modification and suppress cell growth (12). It can increase the expression of genes that are regulated by transcription factors (13). It has been indicated that the HDACi increases both the specific productivity and mRNA transcription level in stable CHO cell lines. Furthermore, no cellular toxicity was reported with VPA compared with other widely used HDACi such as NaBut (14).
Blinatumumab, the most advanced bispecific T-cell engager (BiTE) with dual binding specificities (15), was approved for precursor B-cell acute lymphoblastic leukemia (B-cell ALL) on December 3, 2014 (16). BiTE antibodies can form a transient cytolytic synapse between T cells and the tumor target cells. This leads to discharge of T cells contents and induces tumor cell death (17).
Blinatumomab can redirect T cells toward malignant B cells, and induce cancer cell lysis. The 55 kDa bispecific antibody (BsAb) has an anti-CD3 arm to bind CD3+ T-cells, and an anti-CD19 arm to couple to CD19+ lymphoma cells (15). Preclinical studies illustrated that blinatumomab's efficacy is dependent on the effector-to-target ratio and on the difference between its affinity for both CD19 and CD3 antigens (18). In the present study, we investigated the phiC31 mediated gene integration for expression of a BiTE antibody (Blinatumomab) in CHO-DG44 cells. This is the first study in which phiC31 integrase is used for (BsAb) expression. We compared the effect of the HDAC inhibitors, NaBut and VPA on specific productivity and cell growth in stably transfected cells. The calcein-AM assay was used to determine the cytotoxic activity of the expressed monoclonal antibody (mAb).

Vector construction
The Blinatumomab sequence was cloned into the FC550A-1 (System Biosciences, USA) plasmid by EcoRV restriction enzyme. Subsequently clones containing ligated plasmid were screened with XhoI restriction enzyme. All cloning steps were done according to our previous study (19). 6 × His-tag was introduced at the C-terminus of the construct for further detection and purification.

Antibody purification
Based on our previous study, supernatants of the stable cell pools containing the expressed BsAb were collected to be purified by using nickel nitrilotriacetic acid (Ni-NTA) resin (QIAGEN, USA). (19). The fraction containing the expressed antibody were collected in 1-ml tubes and stored at -20 °C.

Binding activity of expressed BsAb
We used In-Cell ELISA to study the recognition of both CD3 and CD19 antigens by expressed BsAb. NALM-6 and Jurkat cell lines were utilized. A CD19/CD3 negative cell line (SK-BR-3 cell line) was used as control group. The optical density (OD) of the reactions was measured at 450 nm for each well using an ELISA plate reader (Bio-Rad, Hercules, CA) (19,21).

Determination of specific productivity
To calculate the specific productivity of cells, the cell pool of expressed mAb was cultured. 2×10 5 cells were seeded in shaker bottles, and viable cell density and antibody titer were evaluated during five days. Viable cell density was determined using Trypan blue exclusion method.
Specific productivity (Qp) was measured in pg/cell/day by using the following equation (22): Where ΔP shows the change in antibody titers (μg/ml) between the first and last days of the test, n 0 and n t indicate viable cell densities (10 6 cells/ml) in the first and last days, and t shows culture time in days.

Sodium butyrate and valproic acid treatment
To evaluate the effect of NaBut and VPA on BsAb productivity, 1 × 10 6 cell/ml were cultivated

Statistical analysis
Obtained data were statistically analyzed in GraphPad Prism 6 software using the one-way ANOVA and t test (P value ≤ 0.05). That is to say, calcein-AM release assay was performed as technical triplicates and ELISA assay was performed in duplicates.

Construction of the expression plasmid
The bispecific monoclonal antibody, Blinatumomab sequence was successfully cloned into the EcoRVsite of FC550A-1 vector.

Generation of stable cell pools using phiC31 integrase system
In this research, we used the robust phiC31 were tested for BsAb expression by SDS-PAGE and western blotting (Fig 2). The specific bound of  pg/cell/day)). NaBut treatment decreased the cell viability by 33% in comparison with control group (P<0.05), while VPA-treated cells viability was 59% which is significantly higher than NaButtreated cells (P<0.05) (Fig 4).

Calcein-AM release assay
We tested the efficacy of BsAb at effector to target (E/T) ratios ranging from 10:1 to 2.5:1 by using purified, un-stimulated T lymphocytes as effectors and NALM-6 cells as target cells (Fig 5). Thus, at E/T ratios of 10:1 and 5:1, the expressed BsAb was significantly more cytotoxic than 2.5:1 Fig. 4. Influence of NaBut and VPA on cells specific productivity and cell viability. Stable BsAb producer cell line was cultured in the absence and presence of NaBut and VPA. Error bars indicated SD of triplicate measurements.* indicates that the differences of cell viability of NaBut and VPA treatment group were significant in comparison with control group (P < 0.05). ** indicates that the differences of cell specific productivity of NaBut and VPA treatment groups were significant in comparison with control group (P < 0.05). The difference between cell viability of NaBut treatment group and VPA treatment group was also significant (P < 0.05).

Fig. 5. Schematic of tumor cells killing by cytotoxic T cells redirected with an expressed BsAb (Blinatumomab). 4 phases were
involved. The calcein AM assay was used to quantify cell viability and cytotoxicity. Fig. 6. Dose-response curve of expressed mAb. In this experiment PBMCs from 2 donors were used. Purified T cells were incubated with NALM-6 cells at E/T ratios of 10:1, 5:1, 2.5:1 and cell lysis was determined by calcein AM release after 4 h. The test was done in triplicate. The error bars indicate the standard deviation of two groups. The cytotoxicity observed at higher ratios than 2.5:1 was significantly higher at the concentrations higher than 1 ng/ml (P < 0.05). In order to bring down the costs of recombinant proteins production in the biopharmaceutical industry, researchers worked on regulatory principals of growth and survival during the cell culture process (29). One strategy for increasing CHO cell productivity is the addition of HDAC inhibitors, NaBut, and VPA (29,30). Although, the specific productivity of NaBut was slightly more than VPA, the decrease in cell viability was remarkable in NaBut-treated group. Since VPA is an FDA-approved drug and 5-fold less expensive than NaBut, it is suggested as a cost-effective alternative to NaBut for increasing protein expression in CHO cells (24). NaBut has been demonstrated to increase the expression of genes controlled by mammalian promoters such as cytomegalovirus (CMV) and simian virus 40 (SV40) (31), but it could decrease cell growth and lead to cellular apoptosis (32). In one study, VPA induced cell cycle arrest at G1 phase and was considered as an effective chemical reagent to enhance mAb production in recombinant CHO cells (33). Here the effects of two HDAC inhibitors on stable gene expression using CHO-DG44 cells as host were investigated. Although NaBut increases the specific productivity more than VPA, VPA showed less cytotoxic effects on CHO cells. Therefore, VPA could be a more efficient alternative to NaBut in CHO-DG44 cells.
Our findings proved that the expressed BsAb BsAbs have attracted much interest for their specificity, cost, and ease of production. We used phiC31 integrase as an efficient, site-specific, unidirectional integration system for BsAb expression in CHO cells. Our findings confirm that addition of NaBut and VPA to CHO cells stably expressing BsAb, could increase specific productivity. But, VPA was observed to be more efficient and cost effective when compared to NaBut. Moreover, the obtained results of this study demonstrated that the expressed blinatumomab can be useful for enhancing the cytotoxicity of CD3 + Tcells against CD19 + target cells in vitro. Its efficacy depended on E/T ratio, since it was found to be less effective at low E/T ratios.